Purification of intrinsic factor



Unite. grates 3,008,877 PUREFICATIQN (BF TNTRINSIQ FACTGR Kenneth C.Robbins, Chicago, 111., assignor, by mesne assignments, to Armour andCompany, Chicago, Ill., a corporation of Delaware No Drawing. Filed Nov.1, 1957, Star. No. 693,807 7 Claims. (Cl. 167-74) This invention relatesto a method of obtaining highly purified intrinsic factor preparations.It is particularly related to a method of purifying intrinsic factorutilizing diethylaminoethyl cellulose ion exchangers.

It is known that the duodenum and stomach tissues of animals contain asubstance which is of value in the treatment of anemia in humans, andparticularly in the treatatent Q process are dialyzed to remove salt andother crystalloid materials. Following the dialysis procedures, it hasbeen found desirable to clarify the solution by centrifugation.

In the process of the invention intrinsic factor starting material isfirst put in solution. It is desired that this solution be one in whichthe conditions of pH and ionic strength are proper for later steps inthe process hereinafter described. Following the placing of theintrinsic factor material in solution, the solution may be dialyzed andis thereafter clarified by centrifugation. The solution of intrinsicfactor is then contacted with an anion exchange resin in the form of thediethylam-inoethyl ether of cellulose, i.e. DEAE-cellulose.DEAE-cellulose may be prepared according to procedures which are setforth in used to designate the component or components of animalduodenum or stomach tissue which is of value in the treatment of anemicconditions, particularly of the macrocytic type.

Heretofore various intrinsic factor preparations have 7 been utilized inthe treatment of pernicious anemia, but all of them have suffered fromthe defects of requiring the patient to ingest objectionably largequantities of material. For example, only a short time ago the averagedaily requirement of whole hog duodenum by a pernicious anemia patientwas A to /2 pound which is equal to at least 20 to 40 gms. whendesiccated. Recently efforts have been made to improve this situation bypreparing more concentrated intrinsic factor preparations. For example,in US. Patent No. 2,770,570 there is described a method for thepreparation of intrinsic factor which is clinically active in daily oraldoses on the order of as little as 120 mg. In the method of co-pendingpatent application Serial No. 497,401, filed March 28, 1955, now PatentNo. 2,910,405, there is described a method for the preparation ofintrinsic factor which is clinically active in daily oral doses of 35mg. By daily oral dose is meant the minimum amount of intrinsic factorwhich, when administered orally together with 15 ,ug. of vitamin B topatients with Addisonian pernicious anemia in relapse, will produce asatisfactory clinical response. The procedures for this test are set outin the J. Am. Pharm. Assn. (PRACT), 1940, volume 1, p. 53.

It is therefore a general object of this invention to prepare intrinsicfactor preparations of greatly enhanced potency. It is a particularobject of the invention to provide a method for further purifyingintrinsic factor preparations on DEAE-cellulose ion exhangers. Furtherobjects and advantages will appear as the specification proceeds.

The preferred starting material for use in the process of this inventionis an intrinsic factor preparation prepared according to conventionalprocesses such as is illustrated below in Example I. Generally-- thepotency of such preparations is approximately 35 mg. in a daily oraldose. The discussion following will be concerned primarily with theprocessing of such starting materials.

Intrinsic factor materials prepared according to the methods mentionedabove contain considerable dialyzable inert materials. Therefore, suchmaterials in the present the literature, for example, the reaction ofalkaline cellulose with 2-chloro-N,N-diethylethylamine, JACS 76, p. 1711(1954). Prior to contacting the solution with the ion exchanger it isdesirable to equilibrate the ion exchanger to within the pH range offrom about 5 to 8, and preferably within the pH range of from about 5.4to 7.0. It is also desirable that the solution of intrinsic factor be ata pH within the range of from about 5 to 8 and preferably within therange of from 5 .4 to 7.0. I

The ionic strength of the solution of intrinsic factor during theadsorption step is determined by the pH of the solution. Where the pH ofthe solution is about 5 .4 the ionic strength of the solution must bebelow 0.3. Where the pH of the solution is increased (within the rangeof 5 to 8) the ionic strength may also be increased to a maximum ofabout 0.6. At higher ionic strength than about 0.6 the intrinsic factoris not adsorbed on DEAE-cellulose at any pH. The maximum ionic strengthconsonant with intrinsic factor adsorption is therefore seen to varywith the pH of the solution from about 0.3 at pH 5 to 5.4 to about 0.6at pH 7 to 8. Preferably the ionic strength of the solution in theadsorption step should not be above 0.1.

The operable range of ionic strength of the elution solution is alsorelative to the pH of the solution. Where the pH of the eluant is about5.4 the ionic strength must be in excess of 0.3. Where the pH of theeluant is increased the ionic strength must also be increased so that,for example, where the eluant is at a pH of 7.0 the ionic strength mustbe over 0.6 and preferably over 0.8. When eluting at a pH of 5 .4 it ispreferred to employ an ionic strength of about 0.5. All the intrinsicfactor is eluted from DEAE-cellulose by a solution having an ionicstrength of 0.8 or greater where the pH is within the required range offrom 5 to 8. It is preferred to carry out the elution procedure withsuccessive solutions of increasing ionic strength and separatecollection of the various fractions eluted at different ionic strengths.

The eluate from the adsorbate contains purified intrinsic factor alongwith various crystalloid materials. We have therefore found it desirableto dialyze the eluate to remove such crystalloid materials. Followingthe dialysis the eluate may be lyophilized to obtain an intrinsic factorpreparation in a dry state which is active in daily oral doses of aslittle as about 10 mg.

The following detailed examples are set forth to more fully illustratethe invention.

Example I Intrinsic factor which was clinically active in daily oraldoses of'about 35 mg. Was prepared by the addition of two 1. of ice-coldwater to each kg. of fresh frozen ground centrifugation at 4000 rpm. at2 C. for one hour. The extract of intrinsic factor can be concentratedby either lyophilization or in a Mojonnier pot still. The yield afterlyophilization was 30 g. per kg. of pyloric tissue. The concentratehaving a pH of 4 was adjusted to 7% total solids and to a pH of 5.2 with2 N NaOH. A one molar sodium acetate-acetic acid buifer at a pH of about5.2 was added to the concentrate to give an ionic strength of 0.15 molarsodium acetate at a solids concentration of 6% at 2 C. 95% ethanol at 20C. was added to the extract to a final concentration of about 50% at 5C. After standing overnight at 5 C., the precipitate was removed bycentrifugation at 4000 rpm. at a temperature of 5 C. for a period of 30minutes. It was suspended in 4 volumes (precipitate volume) of ice-colddistilled water and stirred overnight at 2 C. The solution of intrinsicfactor prepared as indicated can be lyophilized and was clinicallyactive in the daily oral dose of approximately 35 mg. The yield afterlyophilization is about 5 to 6 g. per kg. of tissue. The clinicalbiological activity of this preparation is fully described in an articlein Proc. Soc. Expe-r. Biol. and Med., vol. 95, page 781 (1957).

If the material prepared as above is dialyzed before lyophilization, theproduct will be active in a daily oral dose of approximately 20 mg.Dialysis of the intrinsic factor prior to lyophilization will result inthe loss of approximately 60% of the solids.

Example I] Intrinsic factor which was effective in daily oral doses ofabout 35 mg. was fractionated on a DEAE-cellulose exchanger usinggradient elution at pH of 7.0. g. of intrinsic factor prepared asillustrated in Example I were dissolved in 300 ml. of 0.005 M phosphatebuffer at a pH of 7.0. The pH of the solution was readjusted to 7.0. Thesolution was then dialyzed against 8 liters of phosphate buffer (0.005M) for 48 hours at 1 C. It was clarified by centrifugation to eliminatea slight precipitate which appeared during dialysis. This supernatantsolution was then passed through a DEAE-cellulose ion exchange column(dimensions 41 x 2.5 c.) which had been equilibrated with 0.005 Mphosphate buffer at a pH of 7.0 and which had 0.5 m. eq. per g. of basicgroups. The column was washed with the buffer and the wash was collectedin fractions 1 through 10. The intrinsic factor adsorbed on the columnwas eluted by continuous introduction into the column of 0.1 molar NaHPO plus 0.5 molar NaCl into a constant volume reservoir containing 100ml. of 0.005 molar phosphate buffer at a pH of 7.0. The volume of eachfraction was 6.0 ml. and the flow rate was 1.5 ml. per minute. Theexperiment was carried out at 25 C. The bulkof the intrinsic factor wasremoved in fractions 42 to 84 comprising fractions in pools A A and AThe fraction pools, A comprising fractions 1 to 10, A comprisingfractions 42 to 46, A comprising fractions 47 through 53 and Acomprising fractions 54 through 84, were each dialyzed against distilledwater at 1 C. for 48 hours and lyophilized. The Weights of the productfrom these fractions were as follows: A 1.50 g., A 0.36 g., A 0.56 g.,A; 1.20 g. The total recovered intrinsic factor therefore amounted to3.62 gm. or approximately 36% of the starting material. The materialwhich was not adsorbed on the column was found to be inactive. original10 g. material are lost on dialysis, the recovered fractions account formost of the non-dialyzable solids. The clinical activity of each of theadsorbed fractions was about 12.5 mg. per U.S.P. unit.

Example III Fraction A; which contained the bulk of the activity of thepreparation of the previous example was rechromatographed on acarboxymethyl cellulose ion exchanger using gradient elution at a pH5.4. Such a procedure Since 60% of the solids of the utilizingcarboxymethyl cellulose is of no value in the purification of intrinsicfactor since the activity appeared both in the unadsorbed and theadsorbed fractions with no purification of the starting material.

Example IV In another similar experiment intrinsic factor prepared asillustrated in Example I was fractionated on a DEAE- cellulose exchangerusing gradient elution at a pH of about 5.4. The fraction was adsorbedat a pH of 5.4 and was collected in a single fraction C In theexperimental procedures 10 g. of intrinsic factor prepared as indi*cated in Example I were dissolved in 250 ml. of 0.01 M acetate buffer ata pH of 5.4. The solution was dialyzed against 4 liters of the acetatebuffer for 48 hours at 1 C.

It was clarified by centrifugation and the supernatant was thereafterpassed through a DEAE-cellulose ion exchange column which wasequilibrated with 0.01 M acetate buffer at a pH of 5 .4. The column waswashed with the buffer and the wash was collected in fractions through15 (each fraction containing 25 ml.). The gradient was begun at fraction15 and produced with 0.01 molar acetate buifer at a pH of 5.4 containing0.5 molar NaCl into a constant volume reservoir containing 200 ml. of0.1 molar acetate buffer at a pH of 5 .4. The volume of each fractionafter fraction 15 was 20 ml. and the flow rate was 1.5 ml. per minute.The experiment was carried out at 25 C.- Fractio-n pools 2 to 13,fraction C and fractions25 through 38, fraction C were dialyzed againstdistilled water at 1 C. for 48 hours and thereafter lyophilized.Fraction C weighed 2.1 g. and fraction C weighed 1.9 g. The results ofthe testing of these materials are sum= marized in Table I. Theunadsorbed fraction C was not assayed since in a similar experimentcarried on a smaller scale, the unadsorbed fraction was inactive. Thecalcu-- lated U.S.P. activity of C is about 9 mg. which indicates thepurification of intrinsic factor of about 1.5 to times over the dialyzedpreparation of Example I. There ap-' pears to be a slight loss in theunits of activity in this procedure. The intrinsic factor (C obtainedafter lyophilization was found by urinary excretion tests to beclinically active in daily oral doses of approximately 9 mg,approximately /3 of the amount required to produce a satisfactoryresponse of any of the previously known preparations of intrinsicfactor. Fraction C was rechromatographed on DEAE-cellulose usinggradient elution at pH 4.7, which is illustrated in the followingExample V.

Example V 500 mg. of intrinsic factor preparation C obtained asindicated in Example IV was dissolved in 10 ml. 0.01 molar acetate at apH of 4.7. The solution was dialyzed in 200 ml. of the acetate bufferfor 48 hours at 1 C. It was then passed through a DEAE-cell-ulose columnwhich had been equilibrated with 0.01 molar acetate buffer at a pH of4.7. The column was washed with the same buffer solution and thegradient was begun at fraction 15 and was produced with a 0.01 molaracetate buifer containing 0.5 molar NaCl. The fraction volume was 6.0ml. and the flow rate 0.75 ml. per minute. The experiment was carriedout at 25 C. Fraction D contains fraction pools 3 through 6, fraction Dcontains fraction pools 7 through 11, fraction D contains fraction pools22 through 24, and fraction D contains fraction pools 25 through 29.Each of these fractions were dialyzed against distilled water at 1 C.for 48 hours and lyophilized. Fraction D was found to weigh 138 mg, Dweighs 54 mg, D weighs 50 mg, and D Weighs mg. The bulk of the activitywas not adsorbed at this pH and the procedure resulted in no increase inactivity in the most potent fraction collected.

All the materials resulting from the experiments described in Examples Ito V were evaluated for clinical potency according to proceduresdescribed in an article entitled Preparation of Partially PurifiedPorcine Intrinsic Factor, K. C. Robbins and Jane Shields, Proc. Soc.Exper. Biol. and Med, vol. 95, pp. 781-785 (1957).

While in the foregoing specification and examples the invention has beendescribed in specific detail, it will be understood that the inventionis susceptible of many variations and modifications, all of which arewithin the spirit and scope of the invention and of the appended claims.

I claim:

1. A process for the purification of an intrinsic factor preparationcomprising the steps of forming an aqueous solution of said intrinsicfactor preparation at a pH of about 5.4 and having an ionic strength offrom about 0.01 to 0.05; dialyzing said solution; clarifying saidsolution by centrifugation; contacting said solution with adiethylaminoethyl ether of cellulose ion exchanger equilibrated at a pHof about 5.4 whereby intrinsic factor is adsorbed from said solution onsaid ion exchanger; eluting said intrinsic factor from said ionexchanger with an aqueous eluant solution having an ionic strength ofabout 0.5; dialyzing the eluate which contains purified intrinsicfactor; and lyophilizing said eluate to obtain intrinsic factor ofenhanced potency.

2. A process for the purification of an intrinsic factor preparationcomprising the steps of forming an aqueous solution of said intrinsicfactor preparation at a pH of about 7.0 and having an ionic strength offrom about 0.01 to 0.05; dialyzing said solution; clarifying saidsolution by centrifugation; contacting said solution with adiethylaminoethyl ether of cellulose ion exchanger equilibrated at a pHof about 7.0 whereby intrinsic factor is adsorbed from said solution onsaid ion exchanger; eluting said intrinsic factor from said ionexchanger with an aqueous eluant solution having an ionic strength ofabout 0.8; dialyzing the eluate which contains purified intrinsicfactor; and lyophilizing said eluate to obtain intrinsic factor ofenhanced potency.

3. In a process for preparing intrinsic factor of enhanced potencywherein the starting material is a clinically active intrinsic factorpreparation, the steps of contacting a solution of intrinsic factorstarting material with a diethylarninoethyl ether of cellulose ionexchanger equilibrated at a pH within the range of from about 5.4 toabout 7.0 whereby intrinsic factor is adsorbed on said diethylaminoethylether of cellulose ion exchanger, said solution being at a pH within therange of from about 5.4 to about 7.0 and having an ionic strength of notmore than about 0.1, and eluting said intrinsic factor from saiddiethylaminoethyl ether of cellulose ion exchanger with an aqueoussolution having an ionic strength within the range of from about 0.3 to1.0 whereby an aqueous solution of intrinsic factor of enhanced potencyis obtained.

4. A process for preparing intrinsic factor of enhanced potency whereinthe starting material is clinically active intrinsic factor comprisingthe steps of forming an aqueous solution of intrinsic factor startingmaterial, adjusting the pH of said solution to approximately 5.4,adjusting the ionic strength of said aqueous solution to less than 0.1,dialyzing said solution against a sodium acetate buffer, clarifying saidsolution by centrifugation, contacting said solution with adiethytlaminoethyl ether of cellulose ion exchanger equilibrated at a pHof about 5.4 whereby the intrinsic factor from said aqueous solution isadsorbed on said on exchanger, eluting said purified intrinsic factorfrom said ion exchanger with an aqueous solution having an ionicstrength of about 0.5, dialyzing said eluate and lyophilizing theresulting solution of purified intrinsic factor.

5. A process for the purification of intrinsic factor comprising thesteps of forming an aqueous solution of intrinsic factor; dialyzing saidsolution; clarifying said solution by centrifugation; contacting saidsolution with a diethylaminoethyl ether of cellulose ion exchangerequilibrated at a pH within the range of from about 5 to 8 whereby theintrinsic factor from said solution is adsorbed on said ion exchanger,said solution being at a pH of from about 5 to 8 and having a maximumionic strength ranging from 0.3 to 0.6 according to the pH of thesolution, said diethylaminoethyl ether of cellulose ion exchanger beingequilibrated at a pH and ionic strength approximately equivalent to thepH and ionic strength of said solution of intrinsic factor; and elutingsaid purified intrinsic factor from said ion exchanger with an aqueoussolution of a salt at an ionic strength of more than 0.3, and dialyzingthe resulting eluate.

6. In a process for the purification of intrinsic factor, the steps ofcontacting a solution of intrinsic factor with a diethylaminoethyl etherof cellulose anion exchanger equilibrated at a pH Within the range offrom about 5 to about 8 whereby intrinsic factor is adsorbed on saidanion exchanger, said solution being at a pH within the range of fromabout 5 to about 8 and having an ionic strength of not more than 0.3,and eluting said intrinsic factor from said anion exchanger with anaqueous eluant solution having an ionic strength in excess of 0.3whereby an aqueous solution of intrinsic factor of enhanced potency isobtained.

7. In a process for the purification of intrinsic factor, the steps ofcontacting a solution of intrinsic factor with a diethylarninoethylether of cellulose anion exchanger equilibrated at a pH within the rangeof from about 5 to about 8 whereby intrinsic factor is adsorbed on saidanion exchanger, said solution being at a pH within the range of fromabout 5 to about 8 and having an ionic strength within the range of from0.01 to 0.05, and eluting said intrinsic factor from said anionexchanger with an aqueous eluant solution having an ionic strength inexcess of 0.3 whereby an aqueous solution of intrinsic factor ofenhanced potency is obtained.

References Cited in the file of this patent UNITED STATES PATENTSRobbins Nov. 13, 1956 OTHER REFERENCES

1. A PROCESS FOR THE PURIFICATION OF AN INTRINSIC FACTOR PREPARATIONCOMPRISING THE STEPS OF FORMING AN AQUEOUS SOLUTION OF SAID INTRINSICFACTOR PREPARATION AT A PH OF ABOUT 5.4 AND HAVING AN IONIC STRENGTH OFFROM ABOUT 0.01 TO 0.05, DIALYZING SAID SOLUTION, CLARIFYING SAIDSOLUTION BY CENTRIFUGATION, CONTACTING SAID SOLUTION WITH ADIETHYLAMINOETHYL ETHER OF CELLULOSE ION EXCHANGER EQUILIBRATED AT A PHOF ABOUT 5.4 WHEREBY INTRINSIC FACTOR IS ADSORBED FROM SAID SOLUTION ONSAID ION EXCHANGER, ELUTING SAID INTRINSIC FACTOR FROM SAID IONEXCHANGER, ELUTAQUEOUS ELUANT SOLUTION HAVING AN IONIC STRENGTH OF ABOUT0.5, DIALYZING THE ELUATE WHICH CONTAINS PURIFIED INTRINSIC FACTOR, ANDLYOPHILIZING SAID ELUATE TO OBTAIN INTRINSIC FACTOR OF ENCHANCEDPOTENCY.